Long afterglow phosphorescence image analysis system, use method and matched detection card

Abstract

The invention provides a long afterglow phosphorescence image analysis system, a use method and a matched detection card. The system is composed of a shell, a power adjusting knob, a display screen, a trigger switch I, a power interface, a CCD camera video capture switch, a CCD camera, a main control board, a detection card socket and an excitation light source. The video capture switch and the power adjusting knob are installed on the upper side of the shell, the CCD camera, the CCD camera video capture switch and the main control board are connected, the CCD camera is arranged in the middle of the shell in a suspended mode, the main control board is arranged in the shell, and the excitation light power adjusting knob is in signal connection with the excitation light source through the main control board. The display screen, the trigger switch I and the trigger switch II are respectively arranged on the side surface of the bottom shell; and the detection card socket is arranged at the bottom of the shell. Signal crosstalk between detection areas is avoided, and the detection sensitivity is further improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection equipment, and particularly relates to a long afterglow phosphorescence image analysis system, a using method and a supporting detection card. Background Art

[0002] The luminescence of ultra-long organic phosphorescent materials is a delayed luminescence phenomenon. In real life, this material is also called luminous powder or long afterglow powder. Its luminescence principle belongs to photoluminescence, that is, when the material is excited by a light source, excitation energy is stored in the excited state. When the excitation light source is turned off, the energy is slowly released in the form of light. In 1996, Matsuzawa et al. reported doping europium (Eu), dysprosium (Dy), etc. into the strontium aluminate (srAl 2 O 4 ) system and found that the afterglow could reach 10 hours, and it had good luminescence efficiency, thermal stability, chemical stability, etc. Since then, this rare earth-doped aluminate long afterglow material has received extensive attention in the business community and has been widely used in fields such as lighting luminescence, information encryption and anti-counterfeiting, safety emergency indication, and biological imaging. The immunochromatography technology has developed rapidly due to its characteristics of rapidity, simplicity, and low detection cost, and has been widely used in medical detection, food quality monitoring, drug detection, and environmental monitoring. Combining the detection system of the characteristics of the luminous night pearl-like and the immunochromatography detection card can achieve rapid detection of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs, etc. with high sensitivity and high specificity.

[0003] Fluorescent immunochromatography technology is a new membrane detection technology based on the specific immune reaction of antigen and antibody. This technology uses a strip-shaped fiber chromatography material fixed with a detection line (coated antibody or coated antigen) and a quality control line (antibody) as the stationary phase, and the test solution as the mobile phase. The fluorescently labeled antibody or antigen is fixed at the connection point, and the analyte moves under chromatographic conditions through capillary action. For macromolecular antigens (proteins, viruses, pathogenic bacteria, etc.) with multiple antigenic determinants, the "sandwich" type double-antibody sandwich immunochromatography method is usually adopted, that is, the analyte first binds to the fluorescently labeled antibody under the action of the mobile phase, and then binds to the coated antibody at the detection line to form a "sandwich" type of double-antibody sandwich. For small molecule antigens (agricultural veterinary drugs, prohibited drugs, etc.) with only a single antigenic epitope, after the small molecule antigen binds to the fluorescently labeled antibody, it is difficult to bind to the coated antibody on the detection line due to steric hindrance. Therefore, competitive immunochromatography is mostly used to detect small molecule analytes with a single antigenic epitope.

[0004] However, the traditional fluorescence immunoassay chromatography detection system makes qualitative judgments by the naked eye or conducts quantitative analysis through a photoelectric conversion system. For negative and weakly positive specimens, due to the limitations of naked-eye observation, it is difficult to make judgments by the naked eye, resulting in false-negative results. At the same time, due to background noise and autofluorescence, the traditional photoelectric conversion device causes errors in the result data. In addition, there is a problem of mutual crosstalk of probe signals in the detection area in the traditional photoelectric detection equipment, resulting in insufficient sensitivity and inaccurate detection results, which not only increases the cost of disease diagnosis but also limits its application in on-site rapid detection, remote areas, etc. Summary of the Invention

[0005] The purpose of the present invention is to solve the defects existing in the above-mentioned prior art, and provide a long-afterglow phosphorescent image analysis system, a using method and a supporting detection card.

[0006] The present invention adopts the following technical solutions: A long-afterglow phosphorescent image analysis system is composed of a housing, a power adjustment knob, a display screen, a trigger switch I, a power supply interface, a CCD camera video capture switch, a CCD camera, a main control board, a detection card socket and an excitation light source. The video capture switch and the power adjustment knob are installed on the upper side of the housing. The CCD camera, the CCD camera video capture switch and the main control board are all connected. The CCD camera is suspended in the middle of the housing. The main control board is placed inside the housing. The power adjustment knob, the power supply interface, the excitation light source and the main control board are all connected. The excitation light power adjustment knob is connected to the excitation light source signal through the main control board. The display screen, the trigger switch I and the trigger switch II are respectively installed on the side of the bottom shell, and they are all connected to the main control board. The detection card socket is installed at the bottom of the housing.

[0007] A using method of a long-afterglow phosphorescent image analysis system is characterized in that when a detection card is inserted into the detection card socket, the excitation light source is turned on by pressing the trigger switch II briefly, the video recording of the CCD camera is started by turning on the CCD camera video capture switch, and at the same time, the excitation power of the excitation light source is adjusted by rotating the power adjustment knob. After pressing the trigger switch I briefly, the main control board performs video frame division, wavelet denoising and image enhancement on the video image data collected by the CCD camera, then inputs the processed image data into the neural network model, and finally the result is displayed through the display screen.

[0008] Furthermore, the supporting detection card is composed of a test strip, an upper card shell and a lower card shell. The upper card shell is snap-connected to the lower card shell. The lower card shell has a side excitation light notch, and the long-afterglow test strip is placed inside the lower card shell.

[0009] Furthermore, the excitation light source is located on the side of the side excitation light notch, and the CCD camera is located above the side excitation light notch. The excitation light source forms a 90-degree angle with the detection window of the test card. When the excitation light source emits light, the light shines into the test card through the side notch. The CCD camera is perpendicular to the detection window of the test card.

[0010] The method for the main control board to process image data includes: Step 1. Perform image and video segmentation. Select video frames from frame 160 to frame 220, accurately covering a duration of 2 seconds. Subsequently, starting from the initial afterglow image after extinction, continuously capture 10 afterglow images. Select 10 afterglow images as a group to record the long afterglow images with a single video; Step 2. Perform wavelet transform on the captured images to effectively reduce background white noise. The process of wavelet transform is to directly decompose the input picture into RGB three channels, perform soft threshold filtering on each of the three channels respectively, and finally recombine the three channels to form the image after wavelet transform denoising; Step 3. Perform image segmentation on the long afterglow image sets at each concentration. Given that the positions of the C line and T line in the designed flow-through immunochromatographic test strip are fixed, these lines remain unchanged in the image. Cropping the phosphorescent image of the fixed area can further reduce image noise interference and improve image quality; Step 4. Expand the data set through image enhancement strategies such as image rotation; Step 5. Train and predict the neural network model with the expanded data set in a ratio of 8:2 for the training set to the test set; Step 6. The display screen outputs the detection result.

[0011] The beneficial effects of the present invention: The water-soluble night pearl-like luminescent material with carboxyl groups obtained by the present invention can be prepared in large quantities by the "one-step method", and the material has low toxicity. It has high sensitivity, good stability of the luminescence signal, and little interference from the external background. Even if there are many fluorescent characteristic matrices in the carrier or sample, it can almost eliminate their influence and has strong anti-fluorescence interference ability.

[0012] The obtained long afterglow test card can achieve rapid, sensitive and quantitative detection of the target protein. At the same time, the phosphorescence immunochromatography image analysis system obtained by the present invention can solve the problem that the traditional immunochromatographic detection system cannot identify weak positive samples. Moreover, the method of image processing avoids the problem of signal crosstalk in the detection area. The application of wavelet transform denoising also reduces background noise, improves the signal-to-noise ratio, and further enhances the detection sensitivity. Thus, it can be seen that the present invention can achieve high-throughput detection while improving the detection sensitivity and has certain application value. Description of the Drawings

[0013] Figure 1Schematic diagram of the detection card structure of the present invention; Figure 2 Spatial position diagram of the detection card, CCD camera module, and excitation light source; Figure 3 Light intensity attenuation diagram of the luminescence effect of the water-soluble long afterglow nanosensor; Figure 4 Graph of the change of the long afterglow test strip image over time; Figure 5 Schematic diagram of the detection system structure; Figure 6 Structural block diagram of the control and processing process of the main control board of the present invention; Figure 7 Confusion matrix of the detection accuracy of the detection system and the classification of some low-concentration samples, where (a) represents the system's six-classification detection model loss value and detection accuracy rate of the five low-concentration samples of 0 、0.01 、0.76 、2.3 、5.2 and 7.6 changing with the number of iterations; (b) represents the confusion matrix result graph obtained by the six-classification test of the system model; Figure 8 Correlation curve graph between the detected concentration of the system and the concentration of clinical samples.

[0014] In the figure, 100 - upper shell of the detection card, 101 - test strip, 102 - lower shell of the detection card, 103 - side excitation light notch, 1003 - detection card; 2001 - shell, 2002 - power adjustment knob, 2003 - display screen, 2004 - trigger switch Ⅰ, 2005 - trigger switch Ⅱ, 2006 - power supply interface, 2007 - CCD camera video capture switch, 2008 - camera, 2009 - main control board, 2010 - detection card socket, 2011 - excitation light source. Detailed implementation manners

[0015] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0016] Embodiment (1) Preparation method of water-soluble long afterglow nano-luminescent material: Add 10 parts of long afterglow nano-material PDBCz, 9,9’-(2,5-dibromo-1,4-phenylene) bis[9H-carbazole], and 2 parts of surfactant polyvinylpyrrolidone (PVP) and mix them; then add 20 mg of carboxymethyl cellulose (CMC) and mix, and add a blend system of water and tetrahydrofuran (volume fraction of water is 85%) thereto, and ultrasonicate it in a water bath environment at 70 °C for 120 minutes using a cell disruptor (working time: 2 s; pause time: 2 s; ultrasonic power: 240 W); next, repeatedly centrifuge and wash the obtained solution and then resuspend it in an appropriate amount of deionized water, and then ultrasonically emulsify it finely using a cell disruptor for 30 minutes (working time: 2 s; pause time: 4 s; ultrasonic power 40 w) to obtain the long afterglow water-soluble nano-luminescent material.

[0017] (2) Preparation method of long afterglow water-soluble nano-luminescent probe: Take 3 mg of the night pearl-like luminescent material from the solution of the above water-soluble night pearl-like luminescent material into a centrifuge tube; and add an appropriate amount of MES solution to a total volume of 1 mL; centrifuge using a refrigerated centrifuge, with the centrifuge working time of 8 minutes, working temperature of 4 °C, and centrifugation speed of 12,000 rmp; then, after centrifugation is completed, pour out the supernatant in the centrifuge tube, and add 1 mL of MES, and ultrasonicate it using a ultrasonic cell disruptor for 1 minute (working time: 2 s; pause time: 2 s; ultrasonic power: 240 W); after ultrasonication, add 1.5 mg of EDC and 1.2 mg of NHS to the solution, and then place the centrifuge tube on a rotary mixer and rotate it for 1 h; after rotating for 1 h, centrifuge using a refrigerated centrifuge (working time of 8 minutes, working temperature of 4 °C, and centrifugation speed of 12,000 rmp), pour out the supernatant after centrifugation, then add 1 mL of PBS, and ultrasonicate it using a ultrasonic cell disruptor for 1 minute (working time: 2 s; pause time: 2 s; ultrasonic power 240 W), then add 100 μL of PBS solution containing 10% BSA, place it on a rotary mixer and rotate it for 1 h; after rotating for 1 h, centrifuge using a refrigerated centrifuge (working time of 8 minutes, working temperature of 4 °C, and centrifugation speed of 12,000 rmp), pour out the supernatant after centrifugation, then add 1 mL of aqueous solution containing 1% BSA, and ultrasonicate it using a ultrasonic cell disruptor for 1 minute (working time: 2 s; pause time: 2 s; ultrasonic power: 240 W) to complete the preparation of the long afterglow water-soluble nano-luminescent probe.

[0018] (3) Preparation method of long afterglow test strip: Use a three-dimensional membrane scribing and gold spraying instrument to respectively aspirate 60 The above solution is evenly sprayed on the glass fiber membrane at intervals of 30 min (the gold spraying interval step size is 2 / cm), and put them in a 37-degree constant temperature box to dry for 4 hours. Then, the absorbent paper and NC membrane were attached to the bottom card respectively. Then, the various antibodies on the test line and the goat anti-mouse antibody on the control line were drawn on the NC membrane using a three-dimensional film spraying instrument, and they were placed in a 37-degree constant temperature box to dry for 4 hours. Finally, the dried glass fiber membrane was attached to the bottom card and cut into 4 mm wide test strips by a CNC cutting machine.

[0019] The long afterglow nanoprobe luminescence characteristic is a luminescence characteristic that gradually decays after being excited. This process can be detected by a highly sensitive CCD camera. Since the luminescence intensity changes from strong to weak, the detection needs to fully capture this light intensity change. In order to capture the self-luminous area of ​​the test strip more clearly, the structure is close to the luminous area. At the same time, because the 365nm light source cannot be illuminated from the side after being close, the matching test card also needs to have a corresponding gap. After the excitation light source is turned off, the luminescence process of the test card is immediately captured. At this time, the luminescence wavelength is 559nm, and the corresponding detection concentration is captured by the camera to form a video data set.

[0020] like Figure 3 and Figure 4 As shown in the figure, after the 365nm excitation light source is turned off, the excited 559nm light shows a gradual attenuation process. The CCD camera captures the continuously dynamically changing afterglow image, and then the MCU performs wavelet transform denoising on the captured video data, and then sends it to the neural network model for detection and classification. Figure 7 As shown in Figure 1, the classification accuracy of weak positive samples and the output representation of the confusion matrix show that the system successfully has a higher detection sensitivity. Figure 8 As shown, there is a linear correlation between the clinical samples and the detection of the phosphorescent immunoassay image analysis system, and it can be seen that the system has a strong correlation with clinical detection.

[0021] A long afterglow phosphorescence image analysis system of the present invention, such as Figure 5As shown in the figure, it is composed of a housing 2001, a power adjustment knob 2002, a display screen 2003, a trigger switch I 2004, a trigger switch II 2005, a power supply interface 2006, a CCD camera video capture switch 2007, a CCD camera 2008, a main control board 2009, a test card socket 2010 and an excitation light source 2011. The video capture switch 2007 and the power adjustment knob 2002 are installed on the upper side of the housing 2001. The CCD camera 2008, the CCD camera video capture switch 2007 and the main control board 2009 are all connected. The CCD camera video capture switch 2007 is used to turn on or off the CCD camera 2008. The CCD camera 2008 is suspended in the middle of the housing 2001. The main control board 2009 is placed inside the housing 2001. The power adjustment knob 2002, the power supply interface 2006, the excitation light source 2011 and the main control board 2009 are all connected. The trigger switch II 2005 and the excitation light power adjustment knob 2002 are both connected to the excitation light source 2011 through the main control board 2009 by signal. The display screen 2003, the trigger switch I 2004 and the trigger switch II 2005 are respectively installed on the side of the bottom shell 2001, and they are all connected to the main control board 2009. The test card socket 2010 is installed at the bottom of the housing 2001.

[0022] When a test card 1003 is inserted into the test card socket 2010, press the trigger switch II 2005 briefly to turn on the excitation light source 2011, turn on the CCD camera video capture switch 2007 to start the video recording of the CCD camera 2008. At the same time, rotate the power adjustment knob 2002 to adjust the excitation power of the excitation light source 2011. After pressing the trigger switch I 2004 briefly, the main control board 2009 performs video frame segmentation, wavelet denoising and image enhancement on the video image data collected by the CCD camera 2008. Among them, video frame segmentation is mainly to continuously obtain the afterglow change images frame by frame according to the base number of 30 frames per second from the captured original long afterglow video data set, forming a long afterglow image data set; then directly decompose the original long afterglow image data set into RGB three channels and use soft threshold wavelet transform to achieve image denoising, and then reconstruct the denoised long afterglow image data set; then use image enhancement technologies such as multi-angle image rotation, image scaling, and image flipping to expand the long afterglow image data set, and then input the processed image data set into the neural network model, and finally the result is displayed through the display screen 2003.

[0023] As Figure 1 shown, the test card 1003 is composed of a test strip 101, an upper card shell 100 and a lower card shell 102. The upper card shell 100 is snap-connected to the lower card shell 102. The lower card shell 102 has a side excitation light notch 103, and the previously prepared long afterglow test strip is placed inside the lower card shell 102.

[0024] As Figure 2As shown in the figure, when detecting, after removing other components in the phosphorescence immunoimage detection system, the spatial relationship between the excitation light source 2011 and the CCD camera 2008 is shown. It can be seen from the figure that the excitation light source 2011 is located on the side of the side excitation light notch 1003, and the CCD camera 2008 is located on the upper side of the side excitation light notch 1003.

[0025] In terms of spatial structure, the excitation light source 2011 forms a 90-degree angle with the detection window of the detection card 1003. When the excitation light source 2011 emits light, the light enters through the side notch of the detection card 1003. The CCD camera 2008 is perpendicular to the detection window of the detection card 1003, and the installation position is as close as possible to the excited light point, so that the excited light can enter the detection range of the CCD camera 2008 to the maximum extent.

[0026] Experiment 1 Experiment on the acquisition and detection of the luminescent image of the long afterglow test strip Experiment time: 2024.03.23 Experiment instrument: Long afterglow phosphorescence image analysis system Experiment plan: Verify the correlation between the concentration of human serum amyloid A (SAA) and the concentration of clinical test samples through the analysis of long afterglow image data Experiment data: Data of six clinical test samples from the hospital, with a total of 396 sample numbers Marking information: The SAA antibody conjugated with the long afterglow probe is marked on the T line, and the goat anti-mouse antibody is marked on the C line Table 1 shows the marking information of the system for detecting SAA samples. Table 2 shows the results of the classification of six low-concentration samples by the system and the comparison with clinical tests. The correct rate represents the performance of the system's image detection. It can be seen that the system detection performance is good, and the correct rates are all 97% and above.

[0027]

[0028] Table 1

[0029] Table 2 As Figures 7 - 8 shown, experimental conclusions: 1. The system has a high classification accuracy rate, up to 0.99. 2. The concentration correlation is good, and the correlation coefficient reaches 0.99.

[0030] As Figure 6As shown, after irradiation with a 365nm light source, the CCD camera 2008 starts to capture the afterglow video dataset. Then, the main control board 2009 performs video frame segmentation, image filtering, data enhancement, etc. on the afterglow video dataset, and then inputs it into the neural network for detection. Since it is detecting delayed luminescence, when the 365nm light source irradiates the blood, the endogenous interference light in the blood does not exist, so a higher accuracy can be obtained. At the same time, by adopting the image processing strategy, the problem of inaccurate accuracy caused by the crosstalk of phosphorescence signals in the detection area can be effectively avoided, and after Gaussian white noise filtering using wavelet transform, the accuracy of classification detection is higher.

[0031] In the present invention, the detection card 1003 is excited by a 365nm light source. After irradiating for 5s, the excitation source 2011 is turned off, and then the CCD camera is turned on to continuously collect images of the excited 550nm light source for 2s to form a video set of the concentration to be measured. The main control board 2009 performs frame segmentation on the video images to form an image dataset, then removes background noises such as Gaussian white noise through wavelet transform, and then trains and tests the image datasets of each concentration through the neural network model, and finally outputs the concentration category of the measured value.

[0032] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A long afterglow phosphorescence image analysis system, characterized in that: The utility model is composed of a shell, a power adjustment knob, a display screen, a trigger switch I, a power interface, a CCD camera video capture switch, a CCD camera, a main control board, a detection card socket and an excitation light source. The video capture switch and the power adjustment knob are installed on the upper side of the shell, the CCD camera, the CCD camera video capture switch and the main control board are all connected, the CCD camera is suspended in the middle of the shell, the main control board is placed inside the shell, the power adjustment knob, the power interface, the excitation light source and the main control board are all connected, the excitation light power adjustment knob is connected to the excitation light source signal through the main control board, the display screen and the trigger switch I and the trigger switch II are respectively installed on the sides of the bottom shell, they are all connected to the main control board, and the detection card socket is installed at the bottom of the shell.

2. A method for using a long afterglow phosphorescence image analysis system, characterized in that: When a detection card is inserted into the detection card slot, short press trigger switch II to turn on the excitation light source, turn on the CCD camera video capture switch to start the video recording of the CCD camera, and rotate the power adjustment knob to adjust the excitation power of the excitation light source. After short pressing trigger switch I, the main control board performs video frame segmentation, wavelet denoising, and image enhancement on the video image data collected by the CCD camera, and then inputs the processed image data into the neural network model. Finally, the result is displayed on the display screen.

3. The system according to claim 1, characterized in that The detection card is composed of a test strip, an upper card shell and a lower card shell. The upper card shell is connected with the lower card shell. The lower card shell has a side excitation light notch. The long afterglow test strip is placed in the lower card shell.

4. The system according to claim 1, characterized in that The excitation light source is located on the side of the side excitation light notch, the CCD camera is located on the upper side of the side excitation light notch, the excitation light source is 90 degrees to the detection window of the detection card, when the excitation light source emits light, the light shines in from the side notch of the detection card, and the CCD camera is vertical to the detection window of the detection card.

5. The method for processing image data by a long afterglow phosphorescence image analysis system according to claim 1, characterized in that: include: Step 1. Perform image and video segmentation; The video frames are selected from the 160th frame to the 220th frame, covering exactly 2 seconds, and then, starting from the initial afterglow image after extinguishing, 10 afterglow images are continuously taken, and the 10 afterglow images are selected as a group to record the long afterglow image in a single video; Step 2. Perform wavelet transform on the captured image to effectively reduce the background white noise; The process of wavelet transform is to decompose the input image into three channels of RGB, perform soft threshold filtering on the three channels respectively, and finally reassemble the three channels to form a wavelet transform denoised image; Step 3. Perform image segmentation on the long afterglow image set at each concentration; The positions of C and T lines in the flow immunoassay strip are fixed, so these lines remain unchanged in the image, and the phosphorescence image of the fixed area is cropped to reduce image noise interference and improve image quality; Step 4. Expand the dataset through image enhancement strategies; Step 5. Use the expanded data set to train and predict the neural network model in a ratio of 8:2 between the training set and the test set; Step 6. The display screen outputs the test results.

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